Project Summary This revised proposal (DK081513) focuses on the investigation of serine/threonine phosphorylation of IRS-2 by ERK, mTOR and p70S6K in mediating initial hepatic insulin resistance. Insulin action in the liver plays an important role in maintaining glucose homeostasis. Defects in hepatic insulin signaling contribute to systematic insulin resistance and the chronic hyperglycemia of type 2 diabetes. In the liver, IRS-2 is the primary physiological substrate for the insulin receptor tyrosine kinase;as such, sufficient functionality of IRS-2 is essential for proper hepatic insulin signaling. IRS-2 contains many potential serine/threonine phosphorylation sites which are potentially targeted by diverse serine/threonine kinases. Serine phosphorylation of IRS-1 has been shown to attenuate insulin signaling;however, little is known about IRS-2. Our preliminary studies have confirmed activation of numerous serine/threonine kinases as well as phosphorylation of multiple serine/threonine residues in IRS-2 from the liver of insulin-resistant animals. Among tested handful known kinases for IRS-1, ERK and mTOR/p70S6K were the only kinases that correlated with hepatic insulin resistance, suggesting that these serine kinases are selectively activated in resistant liver. In this proposal, we hypothesize that serine/threonine phosphorylation of IRS-2 by ERK, mTOR and/or p70S6K forms an initial insulin resistant signal in liver and cause hepatic insulin resistance, leading to peripheral insulin resistance. This application is designed to test this hypothesis in liver and in isolated primary hepatocytes. Dynamic changes in the activation of these kinases and their phosphorylation on IRS-2 during the development of insulin resistance will be monitored. Key serine/threonine kinase will be identified and its interactions with the insulin signaling pathway -and IRS-2 in particular- will be investigated using adenoviral and/or siRNA techniques. Unique strategies will also be applied to identify unknown serine/threonine phosphorylation sites in IRS-2 and IRS-2 kinases. The objective of this proposal is to define the initial insulin resistant signal (activation of kinase and phosphorylation of IRS-2) in liver. The results from this investigation should provide a clearer, integrative understanding of the molecular determinants of insulin sensitivity in the liver as well as the whole organism.